Studies on the mechanism of oxidative phosphorylation. Positive cooperativity in ATP synthesis.
Kinetic and nucleotide binding studies have shown that submitochondrial particles from bovine heart possess three exchangeable binding sites for ADP or GDP. In order of decreasing affinity at neutral pH, these sites will be referred to as sites I, II, and III, and their respective dissociation constants as KI, KII, and KIII. In oxidative phosphorylation experiments in the presence of saturating amounts of inorganic phosphate, rapid ATP (or GTP) synthesis occurred only upon ADP (or GDP) binding to site III. The Eadie-Hofstee plots (v/[S] on the ordinate versus v on the abscissa) of the kinetics of ATP (or GTP) synthesis at variable ADP (or GDP) were, therefore, composed of an initial upward phase, indicating positive cooperativity with respect to substrate concentration, followed by a downward phase where rapid product formation took place. These data allowed calculation of KII from the upward phase and KIII (equivalent to apparent Km) from the downward phase. KI was estimated from Scatchard plots of binding data with radiolabeled ADP or GDP. Thus, together with our previous results, these findings have allowed characterization of the process of ATP or GTP synthesis by bovine-heart submitochondrial particles in terms of KI, KII, KIII, and kcat.
Matsuno-Yagi
A
A
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Hatefi
Y
Y
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000096:biosynthesis*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000648:ultrastructure; D010085:Oxidative Phosphorylation; D013367:Submitochondrial Particles / Q000378:metabolism*
D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate